Cancer specific monoclonal antibodies

ABSTRACT

Novel monoclonal antibodies and binding fragments thereof specific to human breast cancer, lung cancer, colon cancer and other cancers. The monoclonal antibody does not bind to the cell surface of normal human tissues. The corresponding cancer-specific antigen or antigens with an apparent molecular weight of 40-52 130-200 kDa and polynucleotides encoding the antigen and the CDR regions of the antibody are also disclosed, as well as methods for diagnosis, prognosis and treatment of human breast cancer. The antibodies have tumor specificity and are useful for therapy, diagnosis, monitoring, detecting and imaging of cancers. The antibody-recognized cancer-specific surface antigens can serve as targets for detecting, diagnosing, inhibiting or killing cancer cells.

This application claims the priority of U.S. Provisional Application No.60/464,350, filed Apr. 22, 2003, the disclosure of which is expresslyincorporated by reference herein.

FIELD OF THE INVENTION

This invention relates to immunology and cancer diagnosis and therapy.More particularly it relates to cancer cell surface specific proteinswith molecular weight of about 40-52 kDa and about 130-200 kDa, andantibodies specific therefor, hybridomas that produce the antibody,immunochemicals comprising and derived from the antibody, and diagnosticmethods that use the antibody. The invention also relates to the use ofthe antibodies alone or in combination with cytotoxic factor(s) intherapeutic methods.

BACKGROUND OF THE INVENTION

The use of antibodies as “magic bullets” to deliver toxins to cancercells was proposed by Paul Ehrlich over a century ago, and the potentialof targeted immunotherapy has since attracted the attention ofgenerations of investigators. In 1975, with the development of thetechnology for producing monoclonal antibodies (MoAbs), (G. Kohler andC. Milstein, 1975, Nature, 256:495-497; see also Herzenberg andMilstein, Handbook of Experimental Immunology, ed. Weir (BlackwellScientific, London), 1979, pp. 25.1-25.7), it seemed that successfulantibody therapy was imminent. Early trials with monoclonal antibodies,however, revealed significant obstacles to their use in cancer therapy.Immune rejection of murine monoclonal antibodies, and low efficiencieswere reported during initial clinical experience (Kwak et al., 1995,Clinical applications of monoclonal antibodies, In: Biologic Therapy ofCancer, Eds. V. T. DeVita, Jr., S. Hellman and S. A. Rosenberg, 2nd Ed.,J. B. Lippincott Co., Philadelphia, Pa., pp. 553-565).

Ideally, antibody for cancer therapy should have a high affinity for itsantigen, and an effective unconjugated antibody should worksynergistically with the host's immune system effector mechanisms.Therapeutic antibodies that induce effector mechanisms such asantibody-dependent cellular cytotoxicity (ADCC) and complement-dependentcytolysis (Waldman et al., 1994, Ann. Oncol, 5 Suppl. 1:13-17) have thepotential to provide targeted cancer therapy that is safe and effectivewithout the use of potentially harmful conjugates such as toxins orradionuclides.

Nearly all monoclonal antibodies recognizing antigens on human cancercells also bind to normal human cells expressing the same antigen(Jurcic et al., 1996, Cancer Chemotherapy and Biological ResponseModifiers Annual, Eds. Pinedo et al.pp. 168-188). This cross-reactivitypotentially compromises therapeutic effectiveness and raises issues oftoxicity, leading to the continued interest in defining antigenictargets that are unique to tumor cells. It is therefore highly desirableto have cancer-specific antigens and prepare cancer specific antibodies.The identification of unique cancer antigens enables the design ofselective immunotherapy for neoplastic diseases. The capacity to utilizea determinant that is exclusively expressed by cancer cells or tumorcells, but that is not present in normal cells and tissues, insures thetargeting and elimination of the neoplastic cells, while insulating theviability and function of normal cells. For general background in thisregard, please see Colcher et al., 1981, Proc. Natl. Acad. Sci.78:3199-3203.

The process that leads to the discovery of unique cancer antigens is,however, long, tedious and elaborate, and entails an exhaustive weedingout of antigens expressed on both cancer or tumor cells and normaltissues (see e.g. U.S. Pat. Nos. 4,172,124 and 4,196,265). This isbecause malignant cells resemble their normal cell counterparts. Cancercells often have “low visibility” to an individual's immune surveillancesystem, due to the fact that the majority of cancer antigens areself-antigens or auto-antigens that are also expressed by normal cells.Frequently, the cancer antigen is identical to the normal antigenalthough it is expressed at higher levels or endowed with a negligiblemutation insufficient for its distinction from the self-antigen.

In spite of the above-mentioned obstacles, the present inventionprovides new and specific monoclonal antibodies which are immunoreactivewith a cancer-specific cell surface antigen and which are useful inimmmunotherapy, diagnostic, imaging, monitoring and screeningmethodologies.

The monoclonal antibody 7C8 aides in the diagnosis, prognosis, andtreatment of human cancers including breast and lung cancers. Theantibody is reactive to only human cancer cells but not to apparentlynormal human tissues.

SUMMARY OF THE INVENTION

The invention generally relates to a monoclonal antibody, or bindingfragment thereof that binds specifically to as antigens present in humanbreast cancer, human bladder cancer, and, human lung cancer. Theantigens are (i) a polypeptides having an apparent molecular weight ofabout 150 kDa as determined by SDS-PAGE under reducing conditions; and(ii) absent from human breast, bladder and, lung tissue cells.

Preferably, the monoclonal antibody, or binding fragment thereof, of thepresent invention is produced by a hybridoma cell line designated “7C8.”

The monoclonal antibody or binding fragment thereof of the invention maybe Fab fragments, F(ab)₂ fragments, Fab′ fragments, F(ab′)₂ fragments,Fd fragments, Fd′ fragments or Fv fragments. It may also be ananti-idiotypic antibody.

Preferably, the antibody of the invention may be labeled with adetectable moiety, such as a fluorophore, a chromophore, a radionuclide,a chemiluminescent agent, a bioluminescent agent and an enzyme.

The present invention also provides a hybridoma cell line which producesa monoclonal antibody which binds specifically to an antigen present inhuman breast cancer, human bladder cancer, and human lung cancer, theantigen being (i) polypeptides having an apparent molecular weight ofabout 40-50, or 130 -200 kDa as determined by SDS-PAGE under reducingconditions; and (ii) is absent from human breast, lung and bladdertissue cells. In a preferred embodiment, the hybridoma cell lineaccording is the 7C8 cell line.

The present invention further provides an antibody-recognized surfaceantigen present in human breast cancer, human lung cancer, and humanbladder cancer, the antigen being (i) polypeptides having an apparentmolecular weight of about 42-50 or 130-200 kDa as determined by SDS-PAGEunder reducing conditions; and (ii) absent from human breast, lung andbladder tissue cells. This antigen is recognized by monoclonal antibodyproduced by the hybridoma cell line 7C8.

In a further embodiment, the present invention provides a method ofinhibiting or killing cancer cells, comprising: providing to a patientin need thereof the monoclonal antibody, or binding fragment thereof ofthe present invention, under conditions and in an amount sufficient forthe binding to the cancer cells, thereby causing inhibition or killingof the cancer cells by the immune cells of the patient. Preferably, themethod is for the treatment of breast cancer, lung cancer, or bladdercancer. The monoclonal antibody is preferably conjugated with acytotoxic moiety, such as a chemotherapeutic agent, a photoactivatedtoxin, or a radioactive agent. Preferably, the cytotoxic moiety may be aRicin A chain.

Also provided is a monoclonal antibody of the invention or a bindingfragment thereof that is bound to a solid matrix.

The present invention further provides a method for localizing cancercells in a patient, comprising: (a) administering to the patient adetectably-labeled monoclonal antibody of the invention, or bindingfragment thereof; (b) allowing the detectably-labeled (e.g.radiolabeled; flurochrome labeled, or enzyme labeled, especially viaELISA) monoclonal antibody, or binding fragment thereof, to bind to thecancer cells within the patient; and (c) determining the location of thelabeled monoclonal antibody or binding fragment thereof, within thepatient. Also provided is a method of detecting the presence and extentof cancer in a patient, comprising: determining the level of the antigenin a sample of bodily fluid or a tissue section from the patient andcorrelating the quantity of the antigen with the presence and extent ofthe cancer disease in the patient. In preferred embodiment, the antigenis detected by (1) adding monoclonal antibody 7C8 to the sample ortissue section; (2) adding goat anti-mouse IgG antibody conjugated withperoxidase; (3) fixing with diaminobenzidene and peroxide, and (4)examining the sample or section, wherein reddish brown color indicatesthat the cells bear the antigen. According to the method, theeffectiveness of a cancer treatment may be monitored by periodicallymeasuring changes in the level of the antigen in a body fluid sampletaken from a patient undergoing the therapy, and correlating the changein level of the antigen with the effectiveness of the therapy, wherein alower level of antigen determined at a later time point relative to thelevel of antigen determined at an earlier time point during the courseof therapy indicates effectiveness of the therapy for the cancerdisease.

In another embodiment, the present invention relates to a method ofdiagnosing the presence of cancer in a patient, comprising: (a)measuring the levels of the antigen in cells, tissues, or body fluids ofthe patient; and (b) comparing the measured levels of the antigen of (a)with levels of the antigen in cells, tissues, or body fluids from anormal human control, wherein an increase in the measured levels of theantigen in the patient versus the normal control is associated with thepresence of the cancer. Also provided is a method of imaging cancer in apatient, comprising administering to the patient the antibody, whereinthe antibody is detectably labeled with paramagnetic ions or with aradioisotope.

The present invention further provides a pharmaceutical compositioncomprising the monoclonal antibody, or binding fragment thereof,according to the invention, and a pharmaceutically acceptable carrier,excipient, or diluent.

Still further provided are a method for downregulating HER2 receptorlevels on an SK-BR-3 cell, comprising contacting the cell with amonoclonal antibody of the invention, and a method for sensitizing tumorcells to cisplatin or doxorubicin, comprising contacting the monoclonalantibody of the invention to the cell, wherein the antibody specificallybinds to the extracellular domain of a HER2 receptor on the cell.

The present invention further relates to a polynucleotide encoding theantigen of the invention, and a polynucleotide encoding the monoclonalantibody of the invention.

Other aspects of the invention are apparent to those skilled in the artfrom the detailed description and examples below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows immunohistochemical staining of various human tissues usingmAb 7C8.

FIG. 2 shows Western blot analysis of MDA-MB468 with clone 7C8.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention provides monoclonal antibodies and bindingfragments thereof that specifically recognize and bind to a cell surfaceantigen expressed by various human tumor cells or cancer cells. Thesurface antigen are either exclusively present, or highly expressed, onthe cancer cells, but are absent from, or less highly expressed ordisplayed, on developmentally related cells which serve as controls. Thenewly discovered cancer-specific surface antigens provide targets fortherapeutic intervention in these cancers disease, as well as fordiagnostic and cell purification purposes.

The present invention provides new monoclonal antibodies directedagainst cancer-specific antigen(s) expressed on human breast cancercells. The monoclonal antibodies were characterized and found to bindspecifically to antigens on the surface of breast cancer cells. Inparticular, a representative, exemplary monoclonal antibody wasisolated, characterized and found to react specifically with surfaceproteins with apparent MW of about 40-52 or 130-200 KDa as determined bySDS-PAGE under reducing conditions. This MoAb was given the designation7C8.

Another embodiment of the present invention relates to monoclonalantibodies, and binding fragments or portions thereof, which recognizethe foregoing cell surface antigen. Thus, the present inventionencompasses the specific monoclonal antibody 7C8, as well as antibodies,preferably monoclonal antibodies, and their binding fragments, havingspecificity for the above-described antigen present on human breastcancer, lung cancer and bladder cancer cells. Nonlimiting examples ofantibody fragments or antigen bindable fragments that bind to epitopeson the antigen include the following: Fab fragments, F(ab)₂ fragments,Fab′ fragments, fragments produced by F(ab) expression libraries,F(ab′)₂ fragments, Fd fragments, Fd′ fragments and Fv fragments. Theantibodies may be human, or from animals other than humans, preferablymammals, such as rat, mouse, guinea pig, rabbit, goat, sheep, and pig.Preferred are mouse monoclonal antibodies and antigen-binding fragmentsor portions thereof. In addition, chimeric antibodies and hybridantibodies are embraced by the present invention.

In accordance with the present invention, the monoclonal antibodies andbinding fragments thereof may be characterized as those which are 1)produced from the hybridoma cell line 7C8; 2) antibodies that arecapable of binding to the same antigenic determinant as does themonoclonal antibody produced by the hybridoma cell line; 3) bindingfragments of the monoclonal antibody produced by the hybridoma cellline; or 4) binding fragments of a monoclonal antibody capable ofbinding to the same antigenic determinant as does the monoclonalantibody produced by the hybridoma cell lines.

The immunoglobulin isotype of the monoclonal antibodies of the presentinvention is IgG1.

According to the present invention, the monoclonal antibodies recognizespecific cell surface antigens expressed by and present on human breastcancer, human lung cancer and human bladder cancer cells.

The surface antigen or antigens are polypeptide having an apparentmolecular weight of about 40-52 or 130-200 KDa as determined by SDS-PAGEunder reducing conditions. The cancer-specific antigen according to thepresent invention may contain conformational epitopes, whose recognitionby the monoclonal antibody of the present invention may be dependent onthe conformational nature of the antigen being intact and not denatured,degraded, or otherwise adversely affected. The cancer-specific antigenmay be glycosylated, and different degree of glycosylation causes it tomigrate as multiple bands on an SDS-PAGE gel. The antigen may bepost-translationally processed, and both its precursor and processedfragments show up on the Western Blot. Alternatively, the antigen may bedegraded and the degraded fragments retain their reactivity to themonoclonal antibody. Another probability is that the cancer cells havemultiple antigen proteins that shows the same epitope that is recognizedby the monoclonal antibody.

The present invention further provides hybridoma cell lines that producemonoclonal antibodies that specifically bind to the antigen. Methods forpreparing hybridoma cell lines are well known in the art. Accordingly,any technique or protocol that results in the production of homogeneouspopulations of antibody molecules to a specific antigen, preferablymonospecific antibody molecules, e.g., monoclonal antibodies, bycontinuous cell lines in culture may be used. Such techniques include,but are not limited to, the hybridoma technique developed by Kohler andMilstein (1975, supra), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., 1983, Immunology Today, 4:72; Cote et al.,1983, Proc. Nat'l Acad. Sci. USA, 80:2026-2030), as well as the EpsteinBarr Virus (EBV)-hybridoma technique to produce human monoclonalantibodies (Cole et al., 1985, In: Monoclonal Antibodies and CancerTherapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of anyimmunoglobulin class, including IgM, IgG, IgE, IgA and IgD, or anysubclass thereof Hybridoma cells may be cultured in vivo or in vitroaccording to established methods.

In a further aspect, monoclonal antibodies can be produced in germ-freeanimals utilizing the technology described in International PatentApplication No. WO 98/02545. Also suitable for use in the presentinvention are hybrid antibodies, chimeric antibodies and humanizedantibodies (e.g., U.S. Pat. No. 5,585,089 to Queen et al.). Antibodies,such as hybrid or chimeric antibodies having human components, orhumanized antibodies, are more preferable for use in therapies of humandiseases or disorders than xenogenic antibodies, because the human orhumanized antibodies are much less likely than xenogenic antibodies toinduce an immune response, particularly an allergic response, whenintroduced into a human host.

In addition, techniques developed for the production of chimericantibodies (Morrison et al., 1984,.Proc. Natl. Acad. Sci. USA,81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608, Takeda etal., 1985, Nature, 314:452-454) by splicing the genes (see below) from amouse antibody molecule of appropriate antigen specificity together withgenes from a human antibody molecule of appropriate biological activityare suitable for use in the present invention.

Further, according to the present invention, the techniques describedfor the production of single chain antibodies (e.g., U.S. Pat. Nos.5,476,786 and 5,132,405 to Huston; Huston et al., 1988, Proc. Natl.Acad. Sci. USA, 85:5879-5883; U.S. Pat. No. 4,946,778 to Ladner et al.;Bird, 1988, Science, 242:423-426 and Ward et al., 1989, Nature,334:544-546) can be adapted to produce cancer-specific single chainantibodies. Single chain antibodies are formed by linking the heavy andlight immunoglobulin chain fragments of the Fv region via an amino acidbridge, resulting in a single chain polypeptide. Univalent antibodiesare also embraced by the present invention. In addition, techniques forthe construction of Fab expression libraries (Huse et al., 1989,Science, 246:1275-1281) are suitable for use in this invention to allowthe rapid and easy identification of monoclonal antibody Fab fragments,or derivatives or analogs, having an altered, preferably increased,specificity.

Antibody fragments containing the idiotype of the antibody of thepresent invention, can be produced by known techniques (Greenspan andBona, 1993, FASEB J., 7(5):437-444 and Nissinoff, 1991, J. Immunol.,147(8):2429-2438). For example, such fragments include, withoutlimitation, the F(ab)₂ fragment, which can be produced by pepsindigestion of the intact antibody molecule; the Fab′ fragment, which canbe produced by reducing the disulfide bridges of the F(ab′)₂ fragments,and the Fab fragments, which can be generated by treating the intactantibody molecule with the enzyme papain and a reducing agent.

It is also envisioned that antibodies can be elicited in an animal hostby immunization with cancer cell-derived immunogenic components, or canbe formed by in vitro immunization (sensitization) of immune cells. Theantibodies can also be produced in recombinant systems in which theappropriate cell lines are transformed, transfected, infected ortransduced with appropriate antibody-encoding DNA. Alternatively, theantibodies can be constructed by biochemical reconstitution of purifiedheavy and light chains.

Using the aforementioned types of antibodies or fragments, cellsdisplaying the specifically recognized surface antigen(s), or theantigen(s) themselves, or an immunogenic fragment or portion thereof,can be detected in a test sample by chromatography onantibody-conjugated solid-phase matrices or supports (see E. Harlow andD. Lane, 1999, Using Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.), or by immunoassays. Preferred areantibodies that specifically recognize and bind to the antigen(s) of thepresent invention.

Nucleic Acid Molecules Encoding Antibodies of the Invention

The present invention also provides for nucleic acid molecules encodingan antibody of the invention (including molecules comprising, oralternatively consisting of, antibody fragments or variants thereof). Ina specific embodiment, a nucleic acid molecule of the invention encodesan antibody (including molecules comprising, or alternatively.consisting of, antibody fragments or variants thereof), comprising, oralternatively consisting of, a V_(H) domain having an amino acidsequence of any one of the V_(H) domains of a heavy chain expressed by acell line expressing an antibody of the invention, and a V_(L) domainhaving an amino acid sequence of a light chain expressed by an cell lineexpressing an antibody of the invention. Preferably, the cell line is7C8.

In order to isolate the V_(H) and V_(L) domains from the hybridoma celllines, PCR primers including V_(H) or V_(L) nucleotide sequences, may beused to amplify the expressed V_(H) and V_(L) sequences contained intotal RNA or MRNA isolated from hybridoma cell line 7C8. Briefly, RNA isisolated from the cell lines and used as a template for RT-PCR designedto amplify the V_(H) and V_(L) domains of the antibodies expressed bythe 7C8 cell line. Cells may be lyzed and total RNA extracted. cDNA maybe synthesized, according to methods well-known in the art, from 1.5-2.5μg of RNA using reverse transciptase and random hexamer primers. cDNA isthen used as a template for PCR amplification of V_(H) and V_(L)domains. Alternatively, total RNA is isolated and then the first strandof cDNA synthesized by reverse-transcription reaction using the d(T)₁₇primer.

Primers used to amplify V_(H) and V_(L) genes may be obtained by methodswell-known to those skilled in the art. For example, the 5′ primers foramplification of mouse V genes may be synthesized according to Coloma etal., (1992, J. Immunol. Methods 152:89-104), and designed to hybridizeto partially conserved sequences in the leader regions of V_(H) andV_(L). The 3′ primer for V_(L) genes was located at V-C junction ofV_(L). The 3′ primer for V_(H) genes may be designed on conservedsequences located at the boundary of V_(H) and CH1. Alternatively,Vk1FOR and Vk1BACK for V_(L) chain, VH1FOR and VH1BACK for V_(H) chainaccording to Orlandi et al. (1989, Cloning immunoglobulin variabledomains for expression by the polymerase chain reaction. Proc. Natl AcadSci. USA 86, 3833-3837) may be used. The PCR amplification may be in avolume of 100 μl and performed with a typical cycle of denaturation at94° C. for 1 min, annealing at 30-42° C. for 1.5 min, and elongation at72° C. for 1.5 min.

The PCR products may then be cloned using vectors, for example, whichhave a PCR product cloning site consisting of a 5′ and 3′ single Tnucleotide overhang, that is complementary to the overhanging singleadenine nucleotide added onto the 5′ and 3′ end of PCR products by manyDNA polymerases used for PCR reactions. The PCR products may also begel-purified and ligated into cloning vector pBluscript KS (Stratagene)at the EcoRV site. The V_(H) and V_(L) domains can then be sequencedusing conventional methods known in the art. A comparison of thesequences of the V_(H) and V_(L) domains from AR20.5 (Ab1) and AR42.1(Ab2) with the Kabat protein database (Kabat, et al., 1991, Sequence ofProteins of Immunological Interest, 5th ed., NIH publication No.91-3242, U.S. Department of Health and Human Services, Bethesda) may beused to determine the Complementary Determining Region (CDR) sequencesfor the antibody gene.

Sequences encoding the cDNAs or genomic clones for the particularantibodies can be used for transformation of a suitable mammalian ornonmammalian host cells or to generate phage display libraries, forexample. Additionally, polypeptide antibodies of the invention may bechemically synthesized or produced through the use of recombinantexpression systems.

The cloned V_(H) and V_(L) genes may be placed into one or more suitableexpression vectors. By way of non-limiting example, PCR primersincluding V_(H) or V_(L) nucleotide sequences, a restriction site, and aflanking sequence to protect the restriction site may be used to amplifythe V_(H) or V_(L) sequences. Utilizing cloning techniques known tothose of skill in the art, the PCR-amplified V_(H) domains may be clonedinto vectors expressing the appropriate immunoglobulin constant region,e.g., the human IgG1 or IgG4 constant region for V_(H) domains, and thehuman kappa or lambda constant regions for kappa and lambda V_(L)domains, respectively. Preferably, the vectors for expressing the V_(H)or V_(L) domains comprise a promoter suitable to direct expression ofthe heavy and light chains in the chosen expression system, a secretionsignal, a cloning site for the immunoglobulin variable domain,immunoglobulin constant domains, and a selection marker such asneomycin. The V_(H) and V_(L) domains may also be cloned into a singlevector expressing the necessary constant regions. The heavy chainconversion vectors and light chain conversion vectors are thenco-transfected into cell lines to generate stable or transient celllines that express full-length antibodies, e.g., IgG using techniquesknown to those of skill in the art (See, for example, Guo et al., J.Clin. Endocrinol. Metab. 82:925-31 (1997), and Ames et al., J. Immunol.Methods 184:177-86 (1995) which are herein incorporated in theirentireties by reference).

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody is determined, the nucleotide sequence of the antibody maybe manipulated using methods well known in the art for the manipulationof nucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel etal., eds., 1998, Current Protocols in Molecular Biology, John Wiley &Sons, NY, which are both incorporated by reference herein in theirentireties), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains may be inspected to identify the sequencesof the complementarity determining regions (CDRs) by methods that arewell known in the art, e.g., by comparison to known amino acid sequencesof other heavy and light chain variable regions to determine the regionsof sequence hypervariability. Using routine recombinant DNA techniques,one or more of the CDRs may be inserted within framework regions, e.g.,into human framework regions to humanize a non-human antibody, asdescribed supra. The framework regions may be naturally occurring orconsensus framework regions, and preferably human framework regions(see, e.g., Chothia et al., J. Mol. Biol. 278: 457479 (1998) for alisting of human framework regions). Preferably, the polynucleotidegenerated by the combination of the framework regions and CDRs encodesan antibody that specifically binds an antigen of the invention.Preferably, as discussed supra, one or more amino acid substitutions maybe made within the framework regions, and, preferably, the amino acidsubstitutions improve binding of the antibody to its antigen.Additionally, such methods may be used to make amino acid substitutionsor deletions of one or more variable region cysteine residuesparticipating in an intrachain disulfide bond to generate antibodymolecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

For some uses, such as for in vitro affinity maturation of an antibodyof the invention, it may be useful to express the V_(H) and V_(L)domains of the heavy and light chains of one or more antibodies of theinvention as single chain antibodies or Fab fragments in a phage displaylibrary. For example, the cDNAs encoding the V_(H) and V_(L) domains ofone or more antibodies of the invention may be expressed in all possiblecombinations using a phage display library, allowing for the selectionof V_(H)/V_(L) combinations that bind to the cancer-specific antigen(s)of the invention, with preferred binding characteristics such asimproved affinity.

In phage display methods, functional antibody domains are displayed onthe surface of phage particles which carry the polynucleotide sequencesencoding them. In particular, DNA sequences encoding V_(H) and V_(L)domains are amplified from animal cDNA libraries (e.g., human or murinecDNA libraries of lymphoid tissues) or synthetic cDNA libraries. The DNAencoding the V_(H) and V_(L) domains are joined together by an scFvlinker by PCR and cloned into a phagemid vector (e.g., pCANTAB 6 orpComb 3 HSS). The vector is electroporated in E. coli and the E. coli isinfected with a helper phage. Phages used in these methods are typicallyfilamentous phage including fd and M13 and the V_(H) and V_(L) domainsare usually recombinantly fused to either the phage gene III or geneVIII. Phages expressing an antigen binding domain that binds to anantigen of interest can be selected or identified with the antigen,e.g., using labeled antigen or antigen bound or captured to a solidsurface or bead. Examples of phage display methods that can be used tomake the antibodies of the present invention include, but are notlimited to, those disclosed in Brinkman et al., J. Immunol. Methods182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al.,Gene 187 9-18 (1997); and Burton et al., Advances in Immunology57:191-280(1994).

The antibodies of the invention can be produced by any method known inthe art for the synthesis of antibodies, in particular, by chemicalsynthesis, or by intracellular immunization (i.e., intrabodytechnology). Methods of producing antibodies also include hybridomatechnology, EBV transformation, and other methods discussed herein aswell as through the use recombinant DNA technology, as discussed below.

Recombinant expression of an antibody of the invention, or fragment,derivative, variant or analog thereof, (e.g., a heavy or light chain ofan antibody of the invention or a single chain antibody of theinvention), involves construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody-encoding nucleotide sequence are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule of the invention, or a heavy or lightchain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. Thus, the inventionincludes host cells containing a polynucleotide encoding an antibody ofthe invention, or a heavy or light chain thereof, or a single chainantibody of the invention, operably linked to a heterologous promoter.In preferred embodiments for the expression of double-chainedantibodies, vectors encoding both the heavy and light chains may beco-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention. Such host-expression systemsrepresent vehicles by which the antibody molecules of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3, NSO cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as E. coli, and morepreferably, eukaryotic cells, especially for the expression of wholerecombinant antibody molecule, are used for the expression of arecombinant antibody molecule.

Once an antibody molecule of the invention has been produced by ananimal, chemically synthesized, or recombinantly expressed, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigen,Protein A/G chromatography, and sizing column chromatography),centrifugation, differential solubility, or by any other standardtechnique for the purification of proteins. In addition, the antibodiesof the present invention or fragments thereof can be fused toheterologous polypeptide sequences described herein or otherwise knownin the art, to facilitate purification.

The antibodies can be employed to prepare antigen-antibody affinitycolumns, which may be used for the purification of the antigen. Forexample, gel supports or beads can be activated with various chemicalcompounds, e.g., cyanogen bromide, N-hydroxysuccinimide esters, andantibodies can be bound thereto. More particularly and by way ofexample, antibodies can be added to Affigel-10 (Biorad), a gel supportwhich is activated with N-hydroxysuccinimide esters, such that theantibodies form covalent linkages with the agarose gel bead support. Theantibodies are then coupled to the gel via amide bonds with a spacerarm. The remaining activated esters are then quenched with ethanolamineHCl, 1 M, pH 8. The column is washed with water, followed by 0.23 Mglycine HCl, pH 2.6, to remove any non-conjugated antibody or extraneousprotein. The column is then equilibrated in phosphate buffered saline(PBS), (pH 7.3) with appropriate detergent, and the sample materials,i.e., cell culture supernatants or cell extracts, for example,containing the cancer-specific antigens (e.g., prepared usingappropriate membrane solubilizing surfactants) are slowly passed overthe column. The column is washed with PBS/surfactant until the opticaldensity falls to background. The protein is then eluted from the columnwith 0.23 M glycine-HCl, pH 2.6/surfactant. The purified antigens isthen dialyzed against PBS/surfactant.

In another embodiment, the present invention embraces the isolatedcancer cell-specific antigen(s), as described herein and as recognizedand bound by the monoclonal antibody 7C8. In particular, the inventionembraces the approximately 40-52 KDa protein antigen. Further embracedby the present invention are cancer cell-specific antigens comprising anepitope recognized by the monoclonal antibody 7C8. The cell surfaceglycoproteins comprising the recognized conformational epitope aresubstantially not present on normal cells.

For example, the 40-52 kDa antigen of the present invention may beidentified on a preparatory 2-D gel using the 7C8 monoclonal antibody,and purified. The purified protein may then be sequenced with one ofmany well-known and commercially available techniques. The partial aminoacid sequence may be used to design degenerative primers or probes,which can be used to clone from a suitable cancer cell the gene or cDNAthat encodes the antigen.

Another aspect of the present invention relates to therapeutic methodsfor the treatment of cancer patients, particularly individuals afflictedwith cancers involving cells displaying the 40-52 kDa cancer-specificantigen, more particularly, breast cancer, colon cancer, esophaguscancer, liver cancer, lung cancer and bladder cancer cells.

The therapeutic methods encompassed by the present invention involveprimary tumors or cancers, as well as metastases. As an example, amethod for inhibiting or killing cancer cells comprises administering toa patient one or more of the monoclonal antibodies having specificityfor the cancer cells, or a binding fragment thereof, as described above,under conditions sufficient for the binding of the monoclonal antibody,or binding fragment, to tumor or cancer cells in the patient. Thebinding of antibodies, or their binding fragments, to the tumor cells orcancer cells induces the inhibiting or killing of the cells by thepatient's immune cells. The above described method employs theantibodies or their binding fragments without modification, relying onthe binding of the antibodies to the surface of the cancer cells in situto stimulate and induce an immune response and attack by autologousimmune cells thereon.

Such antibody-mediated treatment or therapy may also be accompanied byother treatments that are directed to tumor or cancer cells, forexample, radiation, chemotherapy, and the like, as well as by adjunctivetherapies to enhance the immune system's attack on the opsonized canceror tumor cells following the above-described treatment/therapyprocedure(s).

More specifically, a growth factor, lymphokine, or cytokine may beco-administered with one or more of the monoclonal antibodies, forexample, erythropoietin and/or GM-CSF (granulocyte/macrophagecolony-stimulating factor), to stimulate white blood cells and supportthe immunocompetence status of the patient. In addition, chimeric orfusion antibodies, or other recombinant antibodies of the presentinvention may be used in therapies and treatment. For example, a fusionprotein molecule comprising at least the antigen-binding region of anantibody of the invention joined to at least a functionally active orbioactive portion of a second protein having anti-tumor or cancereffects, e.g., a lymphokine or oncostatin, may be used to treat thecancer, particularly, in vivo. Moreover, a chimeric antibody can beprepared, wherein the antigen binding portion or site is joined to ahuman Fc molecule of an immunoglobulin, e.g., IgG1, to promoteantibody-dependent mediated cytotoxicity or complement-mediatedcytotoxicity. Recombinant techniques and protocols as known andpracticed in the art (e.g., U.S. Pat. No. 4,474,893 to Reading) may beused to construct bispecific or bifunctional chimeric antibodies whereinone of the binding specificities is that of the antibody according tothe present invention.

In another aspect, the present invention comprises therapeutic methodsutilizing the described monoclonal antibodies, or binding fragmentsthereof, to which a cytotoxic agent has been bound, affixed or coupled.The binding of the cytotoxic antibodies or binding fragments thereof, tothe tumor or cancer cells inhibits the growth of the cells and optimallykills the cells. Examples of suitable cytotoxic agents includechemotherapeutic compounds, a drug (e.g., Garnett and Baldwin, 1986,Cancer Res., 46:2407-24112), a prodrug, enzymes, a photoactivated toxin,or a radioactive agent. Cytotoxic agents include, but are not limitedto, ricin A chain, abrin A chain, modeccin A chain, gelonin, melphalan,bleomycin, adriamycin, daunomycin, or pokeweed antiviral proteins (PAP,PAPII, or PAP-S).

One of ordinary skills in the art will realize that there are numerousradionuclides and chemocytotoxic agents that can be coupled tocancer-specific antibodies by well-known techniques and delivered to asite to specifically destroy tumor cells and tissue. (See e.g. U.S. Pat.No. 4,542; and Pastan et al., 1986, Cell, 47:641-648). Examples ofphotoactivated toxins include dihydropyridine- and omega-conotoxin(Schmidt et al., 1991, J. Biol. Chem., 266(27):18025-18033). Nonlimitingexamples of imaging and cytotoxic reagents that are suitable for useinclude ¹²⁵I, ¹²³I, ¹¹¹In (e.g., Sumerdon et al., 1990, Nucl. Med.Biol., 17:247-254), ^(99m)Tc, ³²P, ³H and ¹⁴C; fluorescent labels suchas fluorescein and rhodamine; chemiluminescent labels such as luciferin,and paramagnetic ions for use in magnetic resonance imaging (Lauffer etal., 1991, Magnetic Resonance in Medicine, 22:339-342). Antibodies canbe labeled with such reagents using protocols and techniques known andpracticed in the art. See, for example, Wenzel and Meares,Radioimmunoimaging and Radioimmunotherapy, Elsevier, New York, 1983;Colcer et al., 1986, Meth. Enzymol., 121:802-816; and MonoclonalAntibodies for Cancer Detection and Therapy, Eds. Baldwin et al.,Academic Press, 1985, pp. 303-316, for techniques relating to theradiolabeling of antibodies. Yttrium-90 labeled monoclonal antibodieshave been described for maximizing the dose delivered to the tumor orcancer cells and/or tissue, while limiting toxicity to normal tissues(e.g., Goodwin and Meares, 1997, Cancer Supplement, 80:2675-2680). Othercytotoxic radionuclides including, but not limited to, Copper-67 (⁶⁷Cu),Iodine-131 (¹³¹I) and Rhenium-186 can also be used for labelingmonoclonal antibodies.

The detectable/detecting label used is selected according to the imagingmodality to be used. For example, radioactive labels, such as Indium-111(¹¹¹In), Technetium-99m (^(99m)Tc), or Iodine 131, can be used forplanar scans or for single photon emission computed tomography (SPECT).Also, positron-emitting labels such as Fluorine-19 can be used inpositron emission tomography (PET). Paramagnetic ions, such asGadlinium(III) or Manganese(II) can be used in magnetic resonanceimaging (MRI). The monoclonal antibodies can also be labeled withradio-opaque labels for the visualization of cancer cells afterinjection, for example, by X-ray, CATscan, or MRI. In particular, forlung cancers, localization of the label within the lung, or external tothe lung, permits the determination of the spread of the disease. Theamount of label that is present and detectable within the lung, forexample, allows the determination of the presence or absence of canceror tumor in the lung.

Other covalent and non-covalent modifications of the monoclonalantibodies, or their binding fragments, as described herein are furtherencompassed for use in the present invention. Such modifications aremeant to include agents which are co-administered with, or areadministered subsequent to, the administration of the antibody(ies), orfragments thereof, to induce or stimulate growth inhibition or killingof the cells to which the antibody(ies) or fragments bind. For example,immunotoxins conjugated to monoclonal antibodies have been found to beefficacious in animal models. The conjugation of MoAbs withribosome-inactivating proteins (e.g., ricin A-chain, ricinus agglutinin,or viscumin) or photoinactivating agents has been described (see, e.g.,D. B. Papkovskii et al., 1990, Biomed. Sci., 1(4):401-406). In addition,pokeweed antiviral protein (PAP) has the ability to disruptanti-apoptotic complexes or inhibit protein synthesis within the targetcell, ultimately resulting in the death of the cell. Further, a numberof small molecules that inhibit tyrosine kinases can be specificallytargeted to cancer cells as growth factor conjugates and which can beadministered with the monoclonal antibodies, or fragments thereof,according to the present invention.

In a related embodiment of the present invention, the monoclonalantibodies according to this invention can be used for immunotherapy,either coupled or uncoupled with a therapeutic agent. These therapeuticagents can be coupled either directly or indirectly to the describedmonoclonal antibodies, using techniques routinely practiced in the art.One example of indirect coupling is by the use of a spacer moiety.Spacer moieties, in turn, can be either insoluble or soluble (Dieher etal., 1986, Science, 231:148) and can be selected to enable drug releasefrom the monoclonal antibody molecule at the target site. Examples oftherapeutic agents which can be coupled to the monoclonal antibodies ofthe invention for anti-cancer immunotherapy are drugs, radioisotopes,lectins, and toxins.

The drugs with which can be conjugated to the monoclonal antibodies ofthe present invention include non-proteinaceous as well as proteinaceouscompounds. The term “non-proteinaceous drugs” encompasses compoundsclassically referred to as drugs, for example, mitomycin C,daunorubicin, and vinblastine. The proteinaceous drugs with which themonoclonal antibodies of the invention can be labeled includeimmunomodulators and other biological response modifiers.

The term “biological response modifiers” is meant to encompasssubstances that are involved in modifying the immune response in suchmanner as to enhance the destruction of the antigen-bearing tumor forwhich the monoclonal antibodies of the invention is specific. Examplesof immune response modifiers include such compounds as lymphokines.Lymphokines include tumor necrosis factor, interleukins, e.g., IL1through IL15, lymphotoxin, macrophage activating factor (MAF), migrationinhibition factor (MIF), colony stimulating factor (CSF), andinterferon. Interferons with which the monoclonal antibodies of theinvention can be labeled include alpha-interferon, beta-interferon andgamma-interferon and their subtypes.

In using radioisotopically conjugated monoclonal antibodies of theinvention for immunotherapy, certain isotopes may be more preferablethan others depending on such factors as leukocyte distribution as wellas isotope stability and emission. If desired, the tumor celldistribution can be evaluated by the in vivo diagnostic techniquesdescribed above. Depending on the malignancy, some emitters may bepreferable to others. In general, alpha and beta particle-emittingradioisotopes are preferred in immunotherapy. For example, if an animalhas solid tumor foci, as in a carcinoma, a high energy beta emittercapable of penetrating several millimeters of tissue, such as ⁹⁰Y, maybe preferable. On the other hand, if the malignancy consists of simpletarget cells, as in the case of leukemia, a shorter range, high energyalpha emitter, such as ²¹²Bi, may be preferable. Examples ofradioisotopes which can be bound to the monoclonal antibodies of theinvention for therapeutic purposes are ¹²⁵I, ¹³¹I, ⁹⁰Y, ⁶⁷Cu, ²¹²Bi,²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, and ¹⁸⁸Re.

Lectins are proteins, usually isolated from plant material, which bindto specific sugar moieties. Many lectins are also able to agglutinatecells and stimulate lymphocytes. Ricin is a toxic lectin that has beenused immunotherapeutically. This is preferably accomplished by bindingthe alpha-peptide chain of ricin, which is responsible for toxicity, tothe antibody molecule to enable site specific delivery of the toxiceffect.

Toxins are poisonous substances produced by plants, animals, ormicroorganisms that, in sufficient dose, are often lethal, especially tocells in the vicinity. Diphtheria toxin (DT), a substance produced byCorynebacterium diphtheria, can be used therapeutically. DT consists ofan alpha and beta subunit which under proper conditions can beseparated. The toxic alpha component can be bound to an antibody andused for site specific delivery to a cell bearing an antigen for whichthe monoclonal antibodies of the invention are specific. Othertherapeutic agents which can be coupled to the monoclonal antibodies ofthe invention are known, or can be easily ascertained, by those ofordinary skill in the art.

The labeled or unlabeled monoclonal antibodies of the present inventioncan also be used in combination with therapeutic agents such as thosedescribed above. Especially preferred are therapeutic combinationscomprising the monoclonal antibody of the invention and immunomodulatorsand other biological response modifiers. Thus, for example, themonoclonal antibodies of the invention can be used in combination withalpha-interferon. This treatment method enhances monoclonal antibodytargeting of cancers by increasing the expression of monoclonal antibodyreactive antigen by the cancer cells (Greiner et al., 1987, Science,235:895). Alternatively, the monoclonal antibodies of this invention maybe used, for example, in combination with gamma-interferon to activateand increase the expression of Fc receptors by effector cells, which, inturn, results in an enhanced binding of the monoclonal antibody to theeffector cell and killing of target tumor cells. Those of skill in theart will be able to select from the various biological responsemodifiers to create a desired effector function which enhances theefficacy of the monoclonal antibodies of the invention.

When the monoclonal antibodies of the present invention are used incombination with various therapeutic agents, the administration of themonoclonal antibody and the therapeutic agent usually occurssubstantially contemporaneously. The term “substantiallycontemporaneously” means that the monoclonal antibody and thetherapeutic agent are administered reasonably close together withrespect to time. Usually, it is preferred to administer the therapeuticagent before the monoclonal antibody. For example, the therapeutic agentcan be administered 1 to 6 days before the monoclonal antibody. Theadministration of the therapeutic agent can be daily, or at any otherinterval, depending upon such factors, for example, as the nature of thetumor, the condition of the patient and the half-life of the agent.

Using the monoclonal antibodies of the present invention, it is possibleto design therapies combining all of the characteristics describedherein. In a given situation, it may be desirable to administer atherapeutic agent, or agents, prior to the administration of themonoclonal antibodies of the invention, in combination with effectorcells and the same, or different, therapeutic agent or agents. Forexample, it may be desirable to treat patients with malignant disease byfirst administering gamma-interferon and interleukin-2 daily for 3 to 5days, and on day 5 administer the monoclonal antibody of the inventionin combination with effector cells, as well as gamma-interferon, andinterleukin-2.

It is also possible to utilize liposomes with the monoclonal antibodiesof the present invention in their membranes to specifically deliver theliposome to the area of the tumor expressing SCLC-specific antigens.These liposomes can be produced such that they contain, in addition tomonoclonal antibody, immunotherapeutic agents, such as those describedabove, which would then be released at the tumor site (e.g., Wolff etal., 1984, Biochem. et Biophys. Acta, 802:259).

The dosage ranges for the administration of the monoclonal antibodies ofthe invention are those large enough to produce the desired effect inwhich the symptoms of the malignant disease are ameliorated. The dosageshould not be so large as to cause adverse side effects, such asunwanted cross-reactions, anaphylactic reactions, and the like.Generally, the dosage will vary with the age, condition, sex and extentof disease of the patient and can be determined by one of skill in theart. The dosage can be adjusted by the individual physician in the eventof any complication. Dosage can vary from about 0.1 mg/kg to about 2000mg/kg, preferably about 0.1 mg/kg to about 500 mg/kg, in one or moredose administrations daily, for one or several days.

Generally, when the monoclonal antibodies of the present invention areadministered conjugated with therapeutic agents, lower dosages,comparable to those used for in vivo immunodiagnostic imaging, can beused. The monoclonal antibodies of the invention can be administeredparenterally by injection or by gradual perfusion over time. Themonoclonal antibodies of the invention can be administeredintravenously, intraperitoneally, intramuscularly, subcutaneously,intracavity, or transdermally, alone or in combination with effectorcells. Preparations for parenteral administration include sterileaqueous or non-aqueous solutions, suspensions, and emulsions. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

As mentioned above, anti-idiotypic monoclonal antibodies to theantibodies according to the present invention may be used in therapiesand treatments in active tumor immunization and tumor therapy (See,Larson et al., 1986, “Therapeutic applications of radiolabeledantibodies: Current situation and prospects”, Int. J. Rad. Appl.Instrum., B).

The monoclonal antibodies, or binding fragments thereof, according tothe present invention, may be used to quantitatively or qualitativelydetect the presence of the cancer-specific antigen on cancer cells. Thiscan be achieved, for example, by immunofluorescence techniques employinga fluorescently labeled antibody, coupled with light microscopic, flowcytometric, or fluorometric detection. In addition, the antibodies, orbinding fragments thereof, according to the present invention mayadditionally be employed histologically, as in immunofluorescence,immunoelectron microscopy, or non-immuno assays, for in situ detectionof the cancer-specific antigen on cells, such as for use in monitoring,diagnosing, or detection assays.

In situ detection may be accomplished by removing a histologicalspecimen from a patient, and applying thereto a labeled antibodyaccording to this invention. The antibody, or antigen-binding fragmentthereof, is preferably applied by overlaying the labeled antibody orfragment onto the biological sample. Through the use of such aprocedure, it is possible to determine not only the presence of theantigen, or conserved variants, or peptide fragments, but also itsdistribution in the examined tissue. The ordinarily skilled in the artwill readily recognize that any of a wide variety of histologicalmethods, e.g., staining procedures, can be modified in order to achievesuch in situ detection.

Immunoassay and other assays for the antigen, or conserved variants, orpeptide fragments thereof, typically comprise incubating a sample, suchas a biological fluid, tissue extract, freshly harvested cells, orlysates of cells that have been incubated in cell culture, in thepresence of a detectably-labeled antibody that recognizes the antigen,conserved variants, or peptide fragments thereof, such as thecancer-specific monoclonal antibodies, or binding fragments thereof, ofthe present invention. Thereafter, the bound antibody, or bindingfragment thereof, is detected by a number of techniques well known inthe art.

The biological sample may be brought into contact with, and immobilizedonto, a solid phase support or carrier, such as nitrocellulose, or othersolid support or matrix, which is capable of immobilizing cells, cellparticles, membranes, or soluble proteins. The support may then bewashed with suitable buffers, followed by treatment with thedetectably-labeled antibody. The solid phase support may then be washedwith buffer a second time to remove unbound antibody. The amount ofbound label on the solid support may then be detected by conventionalmeans. Accordingly, in another embodiment of the present invention,compositions are provided comprising the monoclonal antibodies, orbinding fragments thereof, bound to a solid phase support, such asdescribed herein.

By solid phase support or carrier or matrix is meant any support capableof binding an antigen or an antibody. Well-known supports or carriersinclude glass, plastic, nylon wool, polystyrene, polyethylene,polypropylene, dextran, nylon, amylases, films, resins, natural andmodified celluloses, polyacrylamides, agarose, alumina gels, gabbros,and magnetite. The nature of the carrier can be either soluble to someextent, or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration as long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, cylindrical, as in the inside surface of a testtube, or the external surface of a rod. Alternatively, the surface maybe flat, such as a sheet, film, test strip, stick, and the like. Inaddition, the solid support is preferably inert to the reactionconditions for binding and may have reactive groups, or activatedgroups, in order to attach the monoclonal antibody, a binding fragment,or the binding partner of the antibody. The solid phase support may alsobe useful as a chromatographic support, such as the carbohydratepolymers Sepharose, Sephadex, or agarose. Indeed, a large number of suchsupports for binding antibody or antigen are commercially available andknown to those having skill in the art.

The binding activity for a given antibody may be determined bywell-known methods. Those skilled in the art will be able to determineoperative and optimal assay conditions for each determination byemploying routine experimentation.

With respect to the anti-cancer antibodies, numerous ways to detectablylabel such protein molecules are known and practiced in the art. Forexample, one way the antibodies can be detectably labeled is by linkingthe antibody to an enzyme, e.g., for use in an enzyme immunoassay (EIA),(Voller et al., 1978, “The Enzyme Linked Immunosorbent Assay (ELISA)”,Diagnostic Horizons, 2:1-7; Microbiological Associates QuarterlyPublication, Walkersville, Md.; Voller et al., 1978, J. Clin. Pathol.,31:507-520; Butler et al., 1981, Meths. Enzymol., 73:482-523; EnzymeImmunoassay, 1980, (Ed.) Maggio, CRC Press, Boca Raton, Fla.; EnzymeImmunoassay, 1981, (Eds.) E. Ishikawa et al., Kgaku Shoin, Tokyo,Japan). The enzyme that is bound to the antibody reacts with anappropriate substrate, preferably a chromogenic substrate, so as toproduce a chemical moiety which can be detected, for example, byspectrophotometric, fluorometric, or by visual detection means.Nonlimiting examples of enzymes which can be used to detectably labelthe antibodies include malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, ribonuclease, urease,catalase, glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by chromometricmethods, which employ a chromogenic substrate for the enzyme, or byvisual comparison of the extent of enzymatic reaction of a substratecompared with similarly prepared standards or controls.

A variety of other immunoassays may also be used for detection. Forexample, by labeling the antibodies, or binding fragments thereof, witha radioisotope, a radioimmunoassay (RIA) can be used to detectcancer-specific antigens (e.g., Colcher et al., 1981, Cancer Research,41, 1451-1459; Weintraub, “Principles of Radioimmunoassays”, SeventhTraining Course on Radioligand Techniques, The Endocrine Society, March,1986). The radioactive isotope label can be detected by using a gammacounter or a scintillation counter or by radiography.

The antibodies, or their antigen-binding fragments can also be labeledusing a fluorescent compound. When the fluorescently labeled antibody isexposed to light of the proper wavelength, its presence can then bedetected due to fluorescence. Some of the most commonly used fluorescentlabeling compounds include fluorescein isothiocyanate, rhodamine,phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde andfluorescamine. Detectably labeled fluorescence-emitting metals, such as¹⁵²Eu, or others of the lanthanide series, can be used to label theantibodies, or their binding fragments, for subsequent detection. Themetals can be coupled to the antibodies via such metal chelating groupsas diethylenetriaminepentacetic acid (DTPA), orethylenediaminetetraacetic acid (EDTA).

The antibodies can also be detectably labeled by coupling them to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat develops during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds include, withoutlimitation, luminol, isoluminol, theromatic acridinium ester, imidazole,acridinium salt and oxalate ester. Similarly, a bioluminescent compoundmay be used to label the antibodies of the present invention.Bioluminescence is a type of chemiluminescence found in biologicalsystems in which a catalytic protein increases the efficiency of thechemiluminescent reaction. The presence of a bioluminescent protein isdetermined by detecting the presence of luminescence. Usefulbioluminescent labeling compounds include luciferin, luciferase andaequorin.

Another embodiment of the present invention provides diagnostics,diagnostic methods and imaging methods for cancers and tumors using themonoclonal antibodies and binding fragments thereof as described by thepresent invention. The diagnostic uses of the antibodies according tothe present invention embrace primary tumors and cancers, as well asmetastases. Other cancers and tumors bearing the antigen are alsoamenable to these diagnostic and imaging procedures.

A diagnostic method according to the invention comprises administering,introducing, or infusing the monoclonal antibodies or their bindingfragments as described herein, with or without conjugation to adetectable moiety, such as a radioisotope. After administration orinfusion, the antibody or binding fragment binds to the tumor or cancercells, after which the location of the bound antibodies or fragments isdetected. For detectably labeled antibodies or fragments, for example,those labeled with a radioisotope, imaging instrumentation may be usedto identify the location of the agent within the body. For unlabeledantibodies or fragments, a second detectable reagent may beadministered, which locates the bound antibodies or fragments so thatthey can be suitable detected. Similar methods have been employed forother antibodies, and the skilled practitioner will be aware of thevarious methods suitable for imaging the location of detectably boundantibodies or fragments within the body. As a general guidance, about10-1000 μg, preferably about 50-500 μg, more preferably about 100-300μg, most preferably about 200-300 μg of Protein G-purified MoAb areadministered. For mice, for example, using 200 μg MoAb andintraperitoneal (i.p.) administration, MoAb is injected three times aweek for three weeks. For 300 μg MoAb and intraperitoneal (i.p.)administration, MoAb is injected two times a week for three weeks.Applicable doses for humans include about 100-200 mcg/kg, or 350-700mg/m².

It is to be further understood that a cocktail of different monoclonalantibodies, such as a mixture of the specific monoclonal antibodiesdescribed herein, or their binding fragments, may be administered, ifnecessary or desired, for cancer treatment. Indeed, using a mixture ofmonoclonal antibodies, or binding fragments thereof, in a cocktail totarget several antigens, or different epitopes, on cancer cells, is anadvantageous approach, particularly to prevent evasion of tumor cellsand/or cancer cells due to downregulation of one of the antigens.

In another embodiment, the present invention assists in the diagnosis ofcancers and tumors by the identification and measurement of thecancer-specific antibody in body fluids, such as blood, serum, plasma,sputum and the like. For those cancers that express the antigendescribed herein, the ability to detect the antigen provides earlydiagnosis, thereby affording the opportunity for early treatment. Earlydetection is especially important for cancers difficult to diagnose intheir early stages.

Moreover, the level of antigen detected and measured in a body fluidsample such as blood provides a means for monitoring the course oftherapy for the cancer or tumor, including, but not limited to, surgery,chemotherapy, radiation therapy, the therapeutic methods of the presentinvention, and combinations thereof. By correlating the level of theantigen in the body fluid with the severity of disease, the level ofsuch antigen can be used to indicate successful removal of the primarytumor, cancer, and/or metastases, for example, as well as to indicateand/or monitor the effectiveness of other therapies over time. Forexample, a decrease in the level of the cancer or tumor-specific antigenover time indicates a reduced tumor burden in the patient. By contrast,no change, or an increase, in the level of antigen over time indicatesineffectiveness of therapy, or the continued growth of the tumor orcancer.

In a related embodiment, the present invention provides methods fordiagnosing cancers by assaying for changes of levels in thecancer-specific antigen in cells, tissues or body fluids compared withthe levels in cells, tissues, or body fluids, preferably of the sametype, from normal human controls. A change, especially an increase, inlevels of antigen in the patient versus the normal human control isassociated with the presence of cancer. Typically, for a quantitativediagnostic assay, a positive result indicating that the patient beingtested has cancer, is one in which levels of the antigen in or on cells,tissues or body fluid are at least two times higher, and preferablythree to five times higher, or greater, than the levels of the antigensin or on the same cells, tissues, or body fluid of the normal individualas control. Normal controls include a human without cancer and/ornon-cancerous samples from the patient.

Another embodiment of the present invention relates to pharmaceuticalcompositions comprising one or more monoclonal antibodies, or bindingfragments thereof, according to the invention, together with aphysiologically- and/or pharmaceutically-acceptable carrier, excipient,or diluent.

More specifically, the present invention is directed to pharmaceuticalcompositions comprising a monoclonal antibody, or binding fragmentthereof, including the monoclonal antibodies produced from the hybridomacell line 7C8; antibodies that are capable of binding to the sameantigenic determinant as do the monoclonal antibodies produced by thehybridoma cell line 7C8; binding fragments thereof, and bindingfragments of monoclonal antibody capable of binding to the sameantigenic determinant, and a pharmaceutically-acceptable carrier ordiluent. Preferably, the pharmaceutical composition comprises monoclonalantibody 7C8.

Preferably, the antibodies or binding fragments thereof are deliveredparenterally, such as by intravenous, subcutaneous, or intraperitonealadministration, e.g., injection. Suitable buffers, carriers, and othercomponents known in the art can be used in formulating a compositioncomprising the antibody or fragments for suitable shelf-life andcompatibility for the administration. These substances may includeancillary agents such as buffering agents and protein stabilizing agents(e.g., polysaccharides).

More specifically, therapeutic formulations of the antibodies, orbinding fragments thereof, are prepared for storage by mixing theantibodies or their binding fragments, having the desired degree ofpurity, with optional physiologically acceptable carriers, excipients,or stabilizers (Remington's Pharmaceutical Sciences, 17th edition, (Ed.)A. Osol, Mack Publishing Company, Easton, Pa., 1985), in lyophilizedform or in the form of aqueous solutions. Acceptable carriers,excipients or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,and other organic acids; antioxidants including ascorbic acid; lowmolecular weight (less than about 10 amino acid residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as Tween, Pluronics or polyethylene glycol (PEG).

The antibodies, or binding fragments thereof, also may be entrapped inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization (for example, hydroxymethylcellulose orgelatin-microcapsules and poly-[methylmethacylate] microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules), or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

Antibodies or their binding fragments to be used for in vivoadministration must be sterile. This is readily accomplished byfiltration through sterile filtration membranes, prior to or followinglyophilization and reconstitution. The antibodies, or binding fragmentsthereof, ordinarily will be stored in lyophilized form or in solution.

Therapeutic antibody compositions generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.The route of administration of the antibodies, or binding fragmentsthereof, in accordance with the present invention, is in accord withknown methods, e.g., injection or infusion by intravenous,intraperitoneal, intramuscular, intrarterial, subcutaneous,intralesional routes, by aerosol or intranasal routes, or by sustainedrelease systems as noted below. The antibodies, or binding fragmentsthereof, are administered continuously by infusion or by bolusinjection. Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing theprotein, which matrices are in the form of shaped articles, e.g., films,or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al., 1981, J. Biomed. Mater. Res., 15:167-277 andLanger, 1982, Chem. Tech., 12:98-105), or poly(vinylalcohol)],polylactides (U.S. Pat. No. 3,773,919; EP 58,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., 1983,Biopolymers, 22:547-556), non-degradable ethylene-vinyl acetate (Langeret al., supra), degradable lactic acid-glycolic acid copolymers such asthe LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid (EP 133,988).

While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated antibodiesremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in effectiveness. Rationalstrategies can be devised for antibody stabilization depending on themechanism involved. For example, if the aggregation mechanism isdiscovered to be intermolecular S-S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, and developing specificpolymer matrix compositions.

Sustained-release antibody compositions also include liposomallyentrapped antibodies, or their binding fragments. Liposomes containingthe antibodies are prepared by known methods, for example, DE 3,218,121;Epstein et al., 1985, Proc. Natl. Acad. Sci. USA, 82:3688-3692; Hwang etal., 1980, Proc. Natl. Acad. Sci. USA, 77:4030-4034; EP 52,322; EP36,676; EP 88,046; EP 143,949; EP 142,641; Japanese patent application83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.Ordinarily the liposomes are of the small (about 200-800 Angstroms)unilamellar type in which the lipid content is greater than about 30mol. % cholesterol, the selected proportion being adjusted for theoptimal antibody therapy.

An effective amount of antibody to be employed therapeutically willdepend, for example, upon the therapeutic and treatment objectives, theroute of administration, the age, condition, and body mass of thepatient undergoing treatment or therapy, and auxiliary or adjuvanttherapies being provided to the patient. Accordingly, it will benecessary and routine for the practitioner to titer the dosage andmodify the route of administration, as required, to obtain the optimaltherapeutic effect. A typical daily dosage might range from about 1mg/kg to up to about 100 mg/kg or more, preferably from about 1 to about10 mg/kg/day depending on the above-mentioned factors. Typically, theclinician will administer antibody until a dosage is reached thatachieves the desired effect. The progress of this. therapy is easilymonitored by conventional assays.

Various adjuvants may be used to increase the immunological response tothe antigen and to elicit specific antibodies according to the presentinvention. Depending on the host species to be immunized, adjuvants mayinclude, but are not limited to, Freund's (complete and incomplete),mineral gels, such as aluminum hydroxide, surface active agents, such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanin, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum.

The antibodies of the present invention are also useful for in vitrodiagnostic applications for the detection of cancer cells that possessthe antigen for which the antibodies are specific. As detailed above, invitro diagnostic methods include immunohistological orimmunohistochemical detection of tumor cells (e.g., on human tissue, oron cells dissociated from excised tumor specimens), or serologicaldetection of tumor associated antigens (e.g., in blood samples or otherbiological fluids). Immunohistochemical techniques involve staining abiological specimen, such as a tissue specimen, with one or more of theantibodies of the invention and then detecting the presence on thespecimen of antibody-antigen complexes comprising antibodies bound tothe cognate antigen. The formation of such antibody-antigen complexeswith the specimen indicates the presence of cancer in the tissue.

Detection of the antibody on the specimen can be accomplished usingtechniques known in the art such as immunoenzymatic techniques, e.g.,immunoperoxidase staining technique, or the avidin-biotin technique, orimmunofluorescence techniques (see, e.g., Ciocca et al., 1986,“Immunohistochemical Techniques Using Monoclonal Antibodies”, Meth.Enzymol., 121:562-79 and Introduction to Immunology, Ed. Kimball,(2.sup.nd Ed), Macmillan Publishing Company, 1986, pp. 113-117).Serologic diagnostic techniques involve the detection and quantificationof tumor-associated antigens that have been secreted or “shed” into theserum or other biological fluids of patients thought to be sufferingfrom, as mentioned above. Such antigens can be detected in the bodyfluids using techniques known in the art, such as radioimmunoassays(RIA) or enzyme-linked immunoabsorbant assays (ELISA), wherein antibodyreactive with the shed antigen is used to detect the presence of theantigen in a fluid sample (See, e.g., Uotila et al., 1981, J. Immunol.Methods, 42:11 and Fayed et al., 1998, Disease Markers, 14:155-160).

In yet a further aspect of the invention, monoclonal antibodies orbinding fragments to the antigen are provided labeled with a detectablemoiety, such that they may be packaged and used, for example, in kits,to diagnose or identify cells having the aforementioned antigen. Thekits preferably contain an instruction manual for use of the kit.Non-limiting examples of such labels include fluorophores such asfluorescein isothiocyanate; chromophores, radionuclides, or enzymes.Such labeled antibodies or binding fragments may be used for thehistological localization of the antigen, ELISA, cell sorting, as wellas other immunological techniques for detecting or quantifying theantigen, and cells bearing the antigen, for example.

The antibodies of the present invention exhibit cancer or tumorspecificity. In this regard, MoAb 7C8 reacts with several human cancercells, including breast, lung, and bladder cancers, but not withnon-cancer cells of the same tissues (see Table 2, infra).

Characterization of the cancer-specific antigens shows that they aredetected by MoAb 7C8, but migrate as multiple as a single bands byWestern Blot analysis (FIG. 2).

EXAMPLES

The examples as set forth herein are meant to exemplify the variousaspects of carrying out the invention and are not intended to limit theinvention in any way.

Example 1

Mice were immunized with intact human breast cancer cells. Spleens ofimmunized mice were fused with HL-1 myeloma cells to generate 1,000monoclonal hybridoma cultures. Supernatant fluids from these cultureswere screened in solid phase EIAs for the presence of immunoglobulinreactive with live breast cancer cell lines and not reactive with livenormal human mammary epithelial cells. Whereas many culturesdemonstrated immunoglobulin reactive with all test antigens, clone 7C8contained immunoglobulin reactive only with the breast cancer cells butnot the normal epithelial cells.

Upon further study against human cancer tissue arrays byImmunohistochemistry, monoclonal antibody 7C8 was demonstrated to bereactive and bind to 67% of breast cancer, 42% of lung cancer and 92% ofbladder cancer, but not reactive and bind to corresponding normal humantissues. Monoclonal antibody 7C8 detected multiple protein bands atabout 40-52 and 130-200 kDa in human breast cancer cell line MDA-MB-468cells. The isotype of 7C8 is IgG1.

Materials and Methods

Breast cancer cells: primary cultures were prepared from breast tumorsfrom patients. Tissues were cut into 2˜3 mm pieces and washed inice-cold PBS before digested with collagenase (10 μg/ml) at 37° C. for30 minutes. The digested tissues were disrupted into single cellsuspension in DMEM (containing 4.5 g/L glucose and 110 mg/L sodiumpyruvate and 2 mM L-glutamine) with a 5 ml syringe. The cells were spundown and resuspended in DMEM with 10% FBS (v/v fetal bovine serum) andseeded in tissue culture dishes. After a one-week incubation, the cellswere washed with ice-cold PBS and detached by 10 mM EDTA in PBS. Thecells were collected for immunization.

Immunizations. Four-week old BABL/c mouse was immunized with 10 millionsbreast cancer cells, mixed with an equal volume of complete Freund'sadjuvant. After a few boosting, the titers of tail bleeds from theimmunized mice were tested against live breast cancer cell lines,MDA-MB-468 and MCF-7. Once high titer observed, the spleens were removedfor cell fusion with murine myeloma cells. The hybridoma techniquedescribed originally by Kohler and Milstein, Eur. J. Immunol. 6, 511(1976) has been widely applied to produce hybrid call lines that secretehigh levels of monoclonal antibodies against many specific antigens.

Hybridoma supernatants were tested for presence of antibodies specificfor human breast cancer cell surface specific antigen by ELISA.

For the ELISA, 100,000 cells of either MDA-MB-468, or MCF-7, or MCF-12were seeded in each well of 96-well culture plates in DMEM plus 10% FBS.After a 24-hr incubation, the cells were washed with PBS three times,then incubated with the hybridoma culture supernatants diluted 1:1 inDMEM with 5% FBS for 1 hour at room temp. Plates were washed again and0.1 ml per well of an appropriate dilution of goat anti-mouseimmunoglobulin coupled to horseradish peroxidase was added. The plateswere incubated again for 1 hour at room temperature and then washed asabove. Tetramethylbenzidine (TMB) was added as substrate, incubated for15-20 minutes at room temperature and then the absorbance of each wellwas then read at 492 nm.

Approximately 150 well supernatants reacted with the targets on the cellsurfaces of either one or two, or all three cell lines. Fifty-three (53)hybridomas, including clone 7C8, produced monoclonal antibodies that arehuman breast cancer specific.

Example 2

Immunohistochemistry of Paraffin Sections with monoclonal antibody 7C8:Place the slide in microwave and heat the slide at power 7 for 3.5 min.Incubate sections in three washes of xylene for 5 min each. Incubatesections in two washes of 100% ethanol for 10 min each. Incubatesections in two washes of 95% ethanol for 10 min each. Wash sectionstwice in dH₂O for 5 min each. Wash sections in PBS for 5 min. Forantigen unmasking, incubate the slide in ZBP AgRetriev™ solutionpre-warmed at 37° C. for 30 minutes.

An alternative methods for antigen unmasking is to heat sections in 10mM sodium citrate buffer (pH 6.0) in a microwave oven for I min at fullpower followed by 9 min at medium power. Cool slides for 20 min afterantigen unmasking. Wash sections in dH₂O three times for 5 min each.Incubate sections in 1% hydrogen peroxide for 10 min. Wash sections indH₂O three times for 5 min each. Wash sections in PBS for 5 min. Blockeach section with 100-400 μl blocking solution (5 mg/ml of BSA in PBS)for 1 h at room temperature. Remove solution and add 100-400 μl dilutedprimary antibody to each section (dilute antibody in blocking solution.)Incubate overnight at 4° C. Remove antibody solution and wash sectionsin PBS three times for 5 min each. Add 100-400 μl second antibody,diluted in blocking solution, to each section. Incubate 30 min at roomtemperature. If using the ABC avidin/biotin kit (Vector Laboratories,Burlingame, Calif.) the reagent is prepared according to themanufacturer's instructions and the solution is incubated for 30 min atroom temperature. Remove ABC reagent and wash sections three times inPBS 5 min each. Add 100-400 μl DAB reagent to each section and monitorstaining closely. As soon as the section turns brown, immerse slides indH₂O. Wash sections in dH₂O two times for 5 min each. Document theimages under microscope.

Example 3

Isotyping of Monoclonal Antibodies: Culture supernatants or purifiedantibodies were incubated with the isotyping dip stick manufactured byRoche Molecular Diagnostic Inc.

Example 4

Western Blot Analysis: 10 millions of MDA-MB-468 cells were collected in1 ml of RIPA buffer (50 mM Tris HCI pH 7.4, 1% NP-40, 0.25% sodiumdeoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM sodium orthovanadate, 1 mMNaF, protease inhibitors). 0.1 mg of protein from each sample was usedfor immunoblotting. form of PARP were detected using a mouse monoclonalanti-PARP antibody from Oncogene research. a-Actin was used fornormalizing the loading.

Three different human mammary epithelial cells were seeded at 10,000cells/well of 96-well culture plate. The next day, the live cells wereincubated with 1:1 diluted culture supernatants from hybridoma clone 7C8in 5% BSA in DMEM. MDA-MB-468 cells are estrogen-independent humanbreast cancer cells. Human breast cancer cell line MCF-7 cells areestrogen-dependent, while MCF-12A cells are normal human mammaryepithelial cells. Monoclonal antibody 7C8 reacts to live human breastcancer cell lines MDA-MB-468 and MCF-7 cells but not to normal humanmammary epithelial cell line MCF-12. The results are shown in Table 1.TABLE 1 Immunostaining of Different Breast Epithelial Cells by 7C8 Clone# MDA468 MCF-7B MCF-12A 7C8 0.279 0.139 0.032

Example 5

Immunochemistry staining of different cancer cells with MoAb 7C8: Fixedsections from various human cancer tissues and normal tissues werestained with 0.5 micro-gram/ml of monoclonal antibody 7C8 following theimmunohistochemistry protocol mentioned above. As can be seen from FIG.1, the monoclonal antibody 7C8 reacted to human cancer tissuesspecifically, while no reactivity was observed with the normal humantissues.

A human tissue array of 198 fixed-sections from various human cancertissues or normal tissues were stained with 1 microgram/ml of monoclonalantibody 7C8. As shown in Table 2, monoclonal antibody 7C8 reactedspecifically with 67% of breast cancer tissues, 42% of lung cancers and92% of bladder cancers. No significant staining with normal tissues wereobserved.

Example 6

Characterization of the cancer-specific antigen via Western blotanalysis: 0.1 million cells-equivalent total MDA-MB-468 breast cancercell lysate was loaded in each lane and Western blot with monoclonalantibody clone 7C8 and other monoclonal antibodies at concentration of 1microgram/ml. The results are shown in FIG. 2. Monoclonal antibody 7C8detected multiple bands with an MW of between about 40-52 and 130-200kDa. TABLE 2 Summary of Immunohistochemistry of Tissue Array TissueCancer Normal Breast 8/12 0/6 Lung 5/12 0/6 Bladder 11/12  0/6

The foregoing description and examples have been set forth merely toillustrate the invention and are not intended to be limiting. Sincemodifications of the disclosed embodiments incorporating the spirit andsubstance of the invention may occur to persons skilled in the art, theinvention should be construed broadly to include all variations fallingwithin the scope of the appended claims and equivalents thereof.

1. A monoclonal antibody, or binding fragment thereof, which bindsspecifically to an antigen present in human breast cancer, human lungcancer, and human bladder cancer, the antigen being (i) one or morepolypeptides having an apparent molecular weight of about 40-52 or130-200 kDa as determined by SDS-PAGE under reducing conditions; and(ii) absent from human breast, lung and bladder tissue cells.
 2. Themonoclonal antibody, or binding fragment thereof, according to claim 1,which is produced by a hybridoma cell line designated 7C8 cell line. 3.The monoclonal antibody or binding fragment thereof, according to claim1, wherein the binding fragment is selected from the group consisting ofFab fragments, F(ab)₂ fragments, Fab′ fragments, F(ab′)₂ fragments, Fdfragments, Fd′ fragments and Fv fragments.
 4. An anti-idiotypic antibodywhich mirrors the binding site of the antibody according to claim
 1. 5.A hybridoma cell line which produces a monoclonal antibody which bindsspecifically to an antigen present in human breast cancer, human coloncancer, human esophagus cancer, human liver cancer, human lung cancer,and human ovary cancer, the antigen being (i) one or more polypeptideshaving an apparent molecular weight of about 40-52 or 130-200 kDa asdetermined by SDS-PAGE under reducing conditions; and (ii) it is absentfrom human breast, colon, lung and bladder tissue cells.
 6. Thehybridoma cell line according to claim 5, which is the cell line 7C8. 7.An antibody-recognized surface antigen present in human breast cancer,human colon cancer, human esophagus cancer, human liver cancer, humanlung cancer, and human ovary cancer, the antigen being (i) one or morepolypeptides having an apparent molecular weight of about 150 kDa asdetermined by SDS-PAGE under reducing conditions; and (ii) absent fromhuman breast, lung and bladder tissue cells.
 8. The antibody-recognizedsurface antigen according to claim 7, wherein the antibody that binds tothe antigen is a monoclonal antibody produced by a hybridoma cell linedesignated as 7C8.
 9. A method of inhibiting or killing cancer cells,comprising: providing to a patient in need thereof the monoclonalantibody, or binding fragment thereof, according to claim 1, underconditions and in an amount sufficient for the binding of the monoclonalantibody, or binding fragment thereof, to the cancer cells, therebycausing inhibition or killing of the cancer cells by the immune cells ofthe patient.
 10. The method according to claim 9, wherein the cancer isbreast cancer, colon cancer, esophagus cancer, liver cancer, lungcancer, or ovary cancer
 11. The method according to claim 9, furtherwherein the monoclonal antibody is conjugated with a cytotoxic moiety.12. The method according to claim 11, wherein the cytotoxic moiety is achemotherapeutic agent, a photoactivated toxin, or a radioactive agent.13. The method of claim 11, wherein the cytotoxic moiety is Ricin Achain.
 14. The monoclonal antibody, or binding fragment thereof,according to claim 1, bound to a solid matrix.
 15. A method oflocalizing cancer cells in a patient, comprising: (a) administering tothe patient a detectably-labeled monoclonal antibody, or bindingfragment thereof, according to claim 1; (b) allowing thedetectably-labeled monoclonal antibody, or binding fragment thereof, tobind to the cancer cells within the patient; and (c) determining thelocation of the labeled monoclonal antibody or binding fragment thereof,within the patient.
 16. A method of detecting the presence and extent ofcancer in a patient, comprising: determining the level of the antigenaccording to claim 7 in a sample of bodily fluid or a tissue sectionfrom the patient and correlating the quantity of the antigen with thepresence and extent of the cancer disease in the patient.
 17. The methodaccording to claim 16, wherein the antigen is detected by (1) addingmonoclonal antibody 7C8 to the sample or tissue section; (2) adding goatanti-mouse IgG antibody conjugated with peroxidase; (3) fixing withdiaminobenzidene and peroxide, and (4) examining the sample or section,wherein reddish brown color indicates that the cells bear the antigen.18. A method of monitoring the effectiveness of therapy for cancerdisease, comprising: periodically measuring changes in the level of theantigen according to claim 7 in a body fluid sample taken from a patientundergoing the therapy, and correlating the change in level of theantigen with the effectiveness of the therapy, wherein a lower level ofantigen determined at a later time point relative to the level ofantigen determined at an earlier time point during the course of therapyindicates effectiveness of the therapy for the cancer disease.
 19. Themethod of claim 15, wherein the monoclonal antibody is radiolabeled;flurochrome labeled, or enzyme labeled.
 20. The method of claim 15,wherein the method is an ELISA.
 21. A method of diagnosing the presenceof cancer in a patient, comprising: (a) measuring the levels of theantigen according to claim 7 in cells, tissues, or body fluids of thepatient; and (b) comparing the measured levels of the antigen of (a)with levels of the antigen in cells, tissues, or body fluids from anormal human control, wherein an increase in the measured levels of theantigen in the patient versus the normal control is associated with thepresence of the cancer.
 22. A method of imaging cancer in a patient,comprising administering to the patient the antibody according to claim1, wherein the antibody is detectably labeled with paramagnetic ions orwith a radioisotope.
 23. A pharmaceutical composition comprising themonoclonal antibody, or binding fragment thereof, according to claim 1,and a pharmaceutically acceptable carrier, excipient, or diluent. 24.The monoclonal antibody according to claim 1, labeled with a detectablemoiety.
 25. The monoclonal antibody according to claim 24, wherein thedetectable moiety is selected from the group consisting of afluorophore, a chromophore, a radionuclide, a chemiluminescent agent, abioluminescent agent and an enzyme.
 26. A method for downregulating HER2receptor levels on an SK-BR-3 cell, comprising contacting the cell witha monoclonal antibody of claim
 1. 27. A method for sensitizing tumorcells to cisplatin or doxorubicin, comprising contacting a monoclonalantibody of claim 1 to the cell, wherein the antibody specifically bindsto the extracellular domain of a HER2 receptor on the cell.
 28. Apolynucleotide encoding the antigen of claim
 7. 29. A polynucleotideencoding the monoclonal antibody of claim 1.